Platelet aggregation and release reactions (collectively known as platelet activation) are essential to hemostasis. However, perturbations in platelet mechanisms controlling hemostasis may yield thrombi (blood clots) which are pathogenic when blood flow to dependent tissues is occluded. This is the case in a variety of life-threatening vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion and other blood system thromboses. Therefore, strategies to control platelet aggregation and release are desirable in the treatment of these diseases [L. A. Harker and M. Gent, "The Use of Agents that Modify Platelet Function in the Management of Thrombotic Disorders" in Hemostasis and Thrombosis, R. W. Colman et al., eds., pp. 1438-56, J. B. Lippincott, Co., Philadelphia, Pa. (1987)]. Furthermore, inhibition of platelet aggregation may also be desirable in the case of extracorporeal treatment of blood, such as in dialysis, storage of platelets in platelet concentrates and following vascular surgery.
A large number of compounds, both naturally occurring and synthetic, are known to cause platelet aggregation and release. These include ADP, collagen, arachidonic acid, epinephrine, thrombin, ristocetin, and the thromboxane A.sub.2 mimetic, U46619. The mechanism by which each of these compounds causes platelet aggregation or release varies and involves one of several different receptors on the platelet surface.
A wide variety of antiplatelet agents are currently used for prophylaxis and treatment of arterial thrombotic disorders. Because most of these agents are specific for particular platelet aggregation and/or secretion mechanisms, the agent of choice in a given regimen depends upon the particular mode of platelet activation sought to be inhibited. Antiplatelet agents act in a wide variety of ways, including inhibition of platelet cyclooxygenase, antagonism of the thromboxane A.sub.2 receptor, inhibition of thromboxane A.sub.2 synthetase, elevation of cAMP levels, and antagonism and neutralization of platelet surface glycoprotein IIb/IIIa.
Glycoprotein IIb/IIIa is the platelet fibrinogen receptor. It self-associates as a two-chain complex in a calcium-dependent manner, upon stimulation of platelets with ADP, epinephrine, thrombin or prostaglandin derivatives and precursors thereof [S. J. Shattil et al., "Changes in the Platelet Membrane Glycoprotein IIb/IIIa Complex During Platelet Activation", J. Biol. Chem., 260, pp. 11107-14 (1985); G. A. Margeurie et al., "Human Platelets Possess an Inducible and Saturable Receptor Specific for Fibrinogen", J. Biol. Chem., 254, pp. 5357-63 (1979)]. This results in platelet aggregation mediated by a cross-linking between fibrinogen and the activated glycoprotein IIb/IIIa complexes of two platelets. Specifically, the glycoprotein IIb/IIIa binds to an Arg-Gly-Asp sequence in fibrinogen [M. D. Pierschbacher and E. Ruoslahti, "Cell Attachment Activity of Fibronectin Can Be Duplicated By Small Synthetic Fragments of the Molecule", Nature, 309, pp. 30-33 (1984); K. M. Yamada and D. W. Kennedy, "Dualistic Nature of Adhesive Protein Function: Fibronectin and Its Biologically Active Peptide Fragments Can Autoinhibit Fibronectin Function", J. Cell Biol., 99, pp. 29-36 (1984); N. Ginsberg et al., "Inhibition of Fibronectin Binding to Platelets By Proteolytic Fragments and Synthetic Peptides Which Support Fibroblast Adhesion", J. Biol. Chem., 260, pp. 3931-36 (1985); E. F. Plow et al., "The Effect of Arg-Gly-Asp-Containing Peptides on Fibrinogen and Von Willebrand Factor Binding to Platelets", Proc. Nat. Acad. Sci. USA, 82, pp. 8057-61 (1985); T. K. Gartner and J. S. Bennett, "The Tetrapeptide Analogue of the Cell Attachment Site of Fibronectin Inhibits Platelet Aggregation and Fibrinogen Binding to Activated Platelets", J. Biol. Chem., 260, pp. 11891-94 (1985); M. Kloczewiak et al., "Localization of a Site Interacting With Human Platelet Receptor on Carboxy-Terminal Segment of Human Fibrinogen Gamma Chain", Biochim. Biophys. Res. Comm, 107, pp. 181-87 (1982)].
The most widely used antiplatelet agent is aspirin, a cyclooxygenase inhibitor. Although aspirin blocks ADP- and collagen-induced platelet aggregation, it fails to prevent cyclooxygenase-independent platelet aggregation initiated by agonists, such as thrombin. Moreover, numerous clinical studies have failed to demonstrate a significant benefit of aspirin in the treatment of arterial thrombosis [L. A. Harker and M. Gent, supra]. In addition, aspirin causes a modification of platelet enzymes that requires at least one week to reverse--effectively putting a treated patient at risk for hemorrhaging if surgery or severe trauma should occur during that one week period.
Specific inhibitors of glycoprotein IIb/IIIa, such as monoclonal antibodies [J. S. Bennett et al., "Inhibition of Fibrinogen Binding to Stimulated Human Platelets By a Monoclonal Antibody", Proc. Natl. Acad. Sci. USA, 80, pp. 2417-21 (1983); R. P. McEver et al., "Identification of Two Structurally and Functionally Distinct Sites on Human Platelet Membrane Glycoprotein IIb/IIIa Using Monoclonal Antibodies", J. Biol. Chem., 258, pp. 5269-75 (1983); B. S. Coller, "A New Murine Monoclonal Antibody Reports An Activation-Dependent Change in the Conformation and/or Microenvironment of the Platelet Glycoprotein IIb/IIIa Complex", J. Clin. Invest., 76, pp. 107-08 (1985)] and small Arg-Gly-Asp-containing peptides [T. K. Gartner and J. S. Bennett, supra], are less toxic, faster acting and have a shorter duration of effect as compared to aspirin. These compounds are effective against a number of different platelet aggregation mechanisms, but not against platelet secretion mechanisms. Both Arg-Gly-Asp peptides and antibodies toward glycoprotein IIb/IIIa have been shown to have antithrombotic efficacy in in vivo models of thrombosis [Y. Cadroy et al., "Potent Antithrombotic Effects of Arg-Gly-Asp-Val (RGDV) Peptide In Vivo", Circulat., Part II, 75, p. II-313 (1988); B. S. Coller et al., "Antithrombotic Effect of a Monoclonal Antibody to the Platelet Glycoprotein IIb/IIIa Receptor in an Experimental Animal Model", Blood, 68, pp. 783-86 (1986); S. R. Hanson et al., "Effects of Monoclonal Antibodies Against the Platelet Glycoprotein IIb/IIIa Complex on Thrombosis and Hemostasis in the Baboon", J. Clin. Invest., 81, pp. 149-58 (1988); T. Yasuda et al., "Monoclonal Antibody Against the Platelet Glycoprotein (GP) IIb/IIIa Receptor Prevents Coronary Artery Reocclusion Following Reperfusing With Recombinant Tissue-Type Plasminogen Activator in Dogs", J. Clin. Invest., 81, pp. 1284-91 (1988); B. S. Coller et al., "Inhibition of Human Platelet Function In Vivo With A Monoclonal Antibody", Annals Int. Med., 109, pp. 635-38 (1988)].
In order to effect inhibition of platelet aggregation, Arg-Gly-Asp-containing peptides must be administered at concentrations greater than 10.sup.-5 M. Such requisite dosages imply limited commercial feasibility of those peptides. Monoclonal antibodies to glycoprotein IIb/IIIa are more potent inhibitors of platelet aggregation, but their synthesis in mouse hybridoma cells poses greater potential immunological complications [S. R. Hanson et al., supra]. In addition, Arg-Gly-Asp peptides and antibodies toward glycoprotein IIb/IIIa fail to block platelet secretion. Therefore, these agents may have a limited effectiveness in vivo due to proaggregating effects of released platelet elements and their subsequent activation of the circulating platelet pool.
Thrombin inhibitors, such as heparin, have also been employed as platelet inhibitors. These compounds inhibit or reduce thrombin-mediated platelet aggregation, but have no effect on platelet activation caused by other mechanisms. Furthermore, heparin is known to cause certain dangerous side effects, such as hemorrhaging and thrombocytopenia.
Many attempts to obtain other, more effective antiplatelet agents have centered around snake venoms. Although most snake venoms are known to contain compounds which activate platelets and cause aggregation and secretion, some have been shown to contain inhibitors of platelet aggregation or platelet release reactions as well. Some of these inhibitors, such as those purified from Agkistrodon rhodostoma or Trimeresurus gramineus are enzymes. These two compounds digest fibrinogen and ADP, respectively [C. Ouyang et al., ".gamma.-Fibrinogenase from Agkistrodon rhodostoma (Malayan Pit Viper) Snake Venom", Toxicon, 21, pp. 25-33 (1983); C. Ouyang and T. F. Huang, "Inhibition of Platelet Aggregation by 5'-Nucleotidase Purified from Trimeresurus gramineus Snake Venom", Toxicon, 21, pp. 491-591 (1983)]. Other venom-derived inhibitors act non-enzymatically by blocking the platelet fibrinogen receptor. These include the proteins carinatin, purified from Echis carinatus and trigramin, purified from Trimeresurus gramineus [C. Ouyang et al., "Characterization of the Platelet Aggregation Inducer and Inhibitor from Echis carinatus Snake Venom", Biochim. Biophys. Acta, 841, pp. 1-7 (1985); T. F. Huang et al., "Trigramin", J. Biol. Chem., 262, pp. 16157-63 (1987)].
Trigramin is the best known of all of the snake venom-derived inhibitors. It is a single polypeptide chain with a molecular weight of 10,000. It appears to act by blocking the association of fibrinogen and glycoprotein IIb/IIIa (Kd=2.1-8.8.times.10.sup.-8 M). This inhibition is effectively competed by monoclonal antibodies to glycoprotein IIb/IIIa, as well as by an Arg-Gly-Asp-Ser tetrapeptide. Sequence analysis of a chymotryptic peptide derived from reduced, S-pyridylethyl trigramin reveals that this protein contains an Arg-Gly-Asp sequence. However, whereas small Arg-Gly-Asp containing peptides are effective only in blocking platelet aggregation at concentrations above 10.sup.-5 M, trigramin is effective at concentrations of about 10.sup.-8 M. It is likely that the increased potency of trigramin stems from an optimized alignment of the Arg-Gly-Asp sequence in three-dimensional space, as well as contributions from other structural determinants in the glycoprotein IIb/IIIa-trigramin complex. Despite its effectiveness, trigramin does not inhibit platelet release reactions, namely serotonin release induced by thrombin or U46619. Therefore, the antiplatelet activity of trigramin in vivo may be impaired due to the proaggregating activities of secreted platelet components, resulting in limited efficacy.
Accordingly, the need still exists for an antiplatelet agent which is free from the drawbacks associated with these conventional compounds. Ideally, this antiplatelet agent should be equally effective in inhibiting all mechanisms of platelet activation and its potency should be such that it can be effectively administered in relatively low doses as compared with dosage regimens using conventional antiplatelet agents.